Summary

A novel function of NF-κB in the development of most ectodermal
appendages, including two types of murine pelage hair follicles, was detected
in a mouse model with suppressed NF-κB activity
(cIκBαΔN). However, the developmental
processes regulated by NF-κB in hair follicles has remained unknown.
Furthermore, the similarity between the phenotypes of
cIκBAΔN mice and mice deficient in Eda A1
(tabby) or its receptor EdaR (downless) raised the issue of
whether in vivo NF-κB regulates or is regulated by these novel TNF
family members. We now demonstrate that epidermal NF-κB activity is
first observed in placodes of primary guard hair follicles at day E14.5, and
that in vivo NF-κB signalling is activated downstream of Eda A1 and
EdaR. Importantly, ectopic signals which activate NF-κB can also
stimulate guard hair placode formation, suggesting a crucial role for
NF-κB in placode development. In downless and
cIκBαΔN mice, placodes start to develop,
but rapidly abort in the absence of EdaR/NF-κB signalling. We show that
NF-κB activation is essential for induction of Shh and cyclin D1
expression and subsequent placode down growth. However, cyclin D1 induction
appears to be indirectly regulated by NF-κB, probably via Shh and Wnt.
The strongly decreased number of hair follicles observed in
cIκBαΔN mice compared with
tabby mice, indicates that additional signals, such as TROY, must
regulate NF-κB activity in specific hair follicle subtypes.

Epidermal appendages, including hair follicles, develop through complex
reciprocal signalling interactions between the ectoderm and the underlying
mesoderm (Hardy, 1992). The
fur coat of mice is composed of four types of pelage hair follicles: the long
guard or tylotrich hairs (2-10%), with large bulbs and sebaceous glands; the
shorter and thinner intermediate awl and auchene (25-30%); and the downy
zigzag hairs (60-70%) (Sundberg,
1994). Pelage hair develops in three consecutive waves, starting
at embryonic stage E14.5 for guard hairs (primary hairs), followed by the
intermediate hairs at E16-E17 and zigzag hair development around birth
(secondary hairs) (for reviews, see
Philpott and Paus, 1998;
Schmidt-Ullrich and Paus,
2005).

Hair follicle development is divided into eight morphologically distinct
stages (Schmidt-Ullrich and Paus,
2005). The earliest visible morphological signs of hair
development are placodes (stage 1), groups of rearranged keratinocytes in the
epidermis, which begin to divide and penetrate into the underlying mesoderm
(stages 1-4), eventually giving rise to different parts of the hair follicle,
such as the outer root sheath (ORS), inner root sheath (IRS), cortex and
matrix (stages 5-8) (Paus et al.,
1999). The crucial mesodermal component underneath the placode,
the dermal papilla, is an important signalling centre for hair follicle
development. Signals known to participate in hair follicle initiation, placode
down growth and subsequent morphogenesis of the various parts of the follicle
include Wnt, Bmp2, Bmp4 and Shh (to name a few), together with their effectorsβ
-catenin/Lef1/Tcf, Smad proteins and patched/Gli2
(Millar, 2002;
Schmidt-Ullrich and Paus,
2005). The precise temporal sequence of most signals during hair
follicle development is still unclear. Moreover, there is considerable
signalling redundancy and the specific signals that direct the development of
a particular hair type also remain unknown.

The distinct developmental processes regulated by NF-κB in hair
follicle development are unknown. Compared with tabby and
downless mice, cIκBαΔN mice
demonstrate clear differences in phenotype severity regarding cusp formation,
and number of teeth and hairs (Ohazama et
al., 2004b; Schmidt-Ullrich et
al., 2001). This suggests that NF-κB is not only regulated
by Eda A1/EdaR in hair follicle and tooth development, but also by other
additional signals. Furthermore, it was not clear to what extent NF-κB
acts downstream or upstream of Eda A1/EdaR, given that the expression of
several members of the TNF ligand and receptor multigene families (e.g.
TNFα, LTβ, LTβ receptor, CD40, etc.) are under control of
NF-κB. Therefore, the aim of the current study was to present a detailed
analysis of the physiological role and regulation of NF-κB in murine
primary and secondary hair follicle development.

By mating tabby and downless mice into
(Igκ)3xcona-lacZ (κGal)
NF-κB reporter mice, we report here that NF-κB activity in vivo is
induced downstream of Eda A1/EdaR. Importantly, NF-κB is not needed to
initiate guard hair follicle placode formation. Although an attempt at hair
follicle development up to pre-placode stage 0/1 takes place, no further
placode down growth occurs in the absence of NF-κB. These conclusions
are further supported by the resumption of NF-κB activity after
treatment with recombinant Eda A1 in explants of E13.5 tabby ×κ
Gal embryos, which restores placode down growth. However,
TNFα, and to a lesser extent PMA, also induce NF-κB activity and
subsequent placode down growth, showing that induction of NF-κB activity
is sufficient for initiating placode down-growth. In addition, we demonstrate
that NF-κB is required for Eda A1/EdaR-mediated induction of Shh and
cyclin D1 expression.

Embryonic skin cultures

Skin biopsies of E13.5 embryos of κGal and tabby×
κGal mice were harvested in PBS under a stereo
microscope. The explants were then cultured for 24 hours on Millipore filters
at 37°C in DMEM, supplemented with 10% FCS, 1 mM sodium pyruvate and 100
units/ml penicillin/streptomycin, using Falcon centre-well organ culture
dishes and fine metal grids (Goodfellow). When indicated, recombinant purified
Fc-Eda A1 or Fc-Eda A2 (Gaide and
Schneider, 2003) (0.1-0.5 μg/ml)
(Mustonen et al., 2004),
TNFα (25 ng/ml) or PMA (200 ng/ml) were added to the culture medium.
After 24 hours of culture, the skin explants were treated either for X-Gal
staining or for whole-mount in situ hybridization as described above.

BrdU incorporation for cell proliferation studies

Pregnant females (E14.5 and E15.5) were injected with 100 μg BrdU
(5-bromo-2-deoxyuridine, Roche)/g body weight. After 3-4 hours, embryos were
removed, fixed in Bouin's and embedded in paraffin wax as described above.
Sections (5 μm) were cut. Sections were dewaxed, rehydrated and then
bleached with 0.3% H2O2 in methanol. DNA fragmentation
was preformed in 1 M HCl at 37°C for 5 minutes. After protein digestion
with 50 μg Proteinase K (Roche) for 5 minutes at 37°C, sections were
refixed in 3.7% formaldehyde for 5 minutes at room temperature. For BrdU
detection, the Vector M.O.M. Immunodetection Kit for peroxidase (Vector
Laboratories, #PK-2200) was used, together with a mouse monoclonal anti-BrdU
antibody (Sigma; Clone BU33). The POD substrate reaction was carried out with
DAB (Sigma). Sections were counterstained with Haematoxylin.

RESULTS

We have previously shown that cIκBαΔN
mice do not develop guard and zigzag pelage hair follicles. Analysis of
IκBαΔN expression, under the control of the β-catenin
locus, in the skin of P0 cIκBαΔN
knock-in mice displayed readily detectable amounts of IκBαΔN
protein (Fig. 1A). This was
expected because β-catenin mRNA and protein is highly expressed in hair
follicles and interfollicular epidermis at any developmental stage, and, thus,
guarantees expression of the super-repressor IκBαΔN at these
sites (data not shown) (Huelsken et al.,
2001). Endogenous IκBα and β-catenin protein
amounts remained unchanged (Fig.
1A). The EMSA confirmed equally reduced binding of p50-p65
complexes for cIκBαΔN and
downless (EdaR-mutant) mice (Fig.
1B). The faint p50-p65 DNA binding activity in wild-type skin is
expected, considering the few cells with active NF-κB in hair follicles,
when compared with interfollicular epidermal keratinocytes without any
detectable NF-κB activity (see below). The residual NF-κB
DNA-binding complexes in cIκBαΔN and
downless mice may account for constitutive activity, independent of
IκBα degradation and of Eda A1/EdaR signalling in the skin. We
detected only p50-p65 (NF-κB) complexes, apart from very prominent p50
homodimer binding in the epidermis. We can conclude that overall heterodimeric
NF-κB activity is reduced in
cIκBαΔN and downless mice.

In NF-κB reporter mice (κGal), we had already observed
that there was no NF-κB activity in the interfollicular epidermis before
and after birth (Schmidt-Ullrich et al.,
2001; Schmidt-Ullrich et al.,
1996). cIκBαΔN mice manifest
neither any hyperproliferation of the skin nor any inflammatory processes,
seen in Ikka–/–,
Relb–/– or
K14-Cre/IkkbFl/Fl mice
(Barton et al., 2000;
Hu et al., 2001;
Pasparakis et al., 2002).
Keratinocyte differentiation was examined and was found to proceed normally in
cIκBαΔN mice
(Fig. 1C). Typical markers for
terminal keratinocyte differentiation (loricrin, involucrin, filaggrin and
spinous layer marker keratin 10) were expressed in patterns indistinguishable
from wild type (Fig. 1C). Thus,
NF-κB activity is not needed for epidermal keratinocyte differentiation.
This is in agreement with IKKα-deficient mice, where terminal
differentiation was blocked independently of NF-κB
(Hu et al., 2001).

Reduced NF-κB activity in skin of
cIκBαΔN and downless mice, but
normal perinatal epidermal keratinocyte differentiation in
cIκBαΔN mice. (A) Total
protein extracts of newborn wild-type and
cIκBαΔN (ΔN) skin were analysed
for IκBαΔN expression in a western blot using an
anti-IκBα antibody (lower panel). Mouse embryonic fibroblasts
isolated from cIκBαΔN mice were used as
a positive control (ΔN MEF, right lane). β-Catenin protein levels
remained unchanged in all extracts (upper panel). ns, non-specific. (B)
EMSA of total skin extracts of newborn wild-type,
cIκBαΔN (ΔN) and downless
(dl) skin. Extracts were treated with specific antibodies against NF-κB
p65, p50 and RelB as indicated, which inhibited (α-p65) or upshifted
(α-p50) the DNA-binding complex. No effect was seen with RelB. Strong
p50 homodimer binding is present in skin extracts. (C) Sagittal
cryosections of E17.5 and P0 wild-type and
cIκBαΔN (ΔN) embryos were
incubated with antibodies to different epidermal differentiation markers (AP
substrate, in red), as indicated above (loricrin, involucrin, filaggrin,
keratin 10). Counterstaining was carried out with Mayer's haemalaun
(blue).

In developing pelage hair follicles NF-κB activity is first
observed in pre-placode stage at E14.5

Because placode formation in cIκBαΔN
mice was interrupted at a very early time point of development, we analysed
NF-κB activity at all stages of normal pelage hair formation
(Fig. 2). Whole-mount and
Technovit plastic sections of E10-P0 embryos of κGal mice
revealed that first NF-κB activity in the epidermis was observed at
E14.5 in an placode-initiating (pre-placode) stage, here defined as stage 0/1,
in guard hair placodes (Fig.
2B). At stage 1 and 2, the activity became restricted to the
proximal part of the placode, which grows downwards to invaginate the
mesenchyme (Fig. 2B). At later
stages of guard hair follicle morphogenesis (>E17) and in all adult
follicles, NF-κB activity is detected in the matrix, cortex, inner root
sheath and the sebaceous gland (Fig.
2B) (Schmidt-Ullrich et al.,
2001). NF-κB activity was also present in a similar
expression pattern in all secondary hair follicle placodes, including awl
hairs, although awl hairs develop in
cIκBαΔN mice. However, in
cIκBαΔN mice, awl hairs do have a
slightly different shape, resulting in an awl/tylotrich intermediate, as was
previously also described for tabby mice
(Falconer, 1952;
Schmidt-Ullrich et al., 2001).
The typical ultrastructure could, thus, be regulated by NF-κB. At
earlier embryonic days (E13.5 or before), NF-κB was seen in endothelial
cells of dermal blood vessels (Fig.
2A,B). At P0 and later, occasional X-Gal staining is also seen in
dermal fibroblasts (data not shown). No NF-κB activity is observed in
the interfollicular epidermis or dermal papilla at any time point
(Fig. 2B). In conclusion,
during hair follicle development NF-κB activity is mainly observed in
the proximal part of pelage hair placodes, indicating a role in proliferation
and down growth of hair placodes.

Formation of guard hair placodes is attempted in
cIκBαΔN and downless mice, but
EdaR and NF-κB are needed for subsequent keratinocyte proliferation and
placode down-growth

NF-κB activity during embryonic vibrissae and hair follicle
development analysed in NF-κB-driven β-Gal reporter mice.
(A) Whole-mount X-Gal staining of E10-13
(Igκ)3xcona-lacZ (κGal)
embryos. At E10-11, NF-κB activity is observed only in somites and
endothelial cells of blood vessels. From E12 onwards, activity is seen in
vibrissae follicles and the rim of the eyelids (see also insets E12 and E13).
(B) Sagittal sections of X-Gal stained embryonic and newborn skin were
performed to analyse the most important stages of murine pelage hair follicle
development. At E13.5, arrows indicate endothelial cells of blood vessels. M,
matrix; Co, pre-cortex; DC, dermal condensate; DP, dermal papilla. Broken line
indicates the boundary between epidermis (Epi) and dermis (Der).

To study further the role of NF-κB in proliferation of placode
keratinocytes, expression of G1 phase cyclin D1, a target gene of NF-κB
in several cell types (Hinz et al.,
1999), and bromodeoxyuridine (BrdU) incorporation (as an S-phase
marker) were analysed. Surprisingly, cyclin D1 was not upregulated until hair
placode developmental stage 1/2 and was highly expressed at germ and peg stage
2-3 at E15.5 (Fig. 3B). At
developmental stage 1, only very weak cyclin D1 expression was detected
(Fig. 3B). Thus, the late
cyclin D1 upregulation does not coincide with the start of Eda
A1/EdaR/NF-κB signalling (stage 0/1, E14). However, Shh upregulation
coincides and co-localizes with cyclin D1 expression at stages 2-3
(Fig. 6B,
Fig. 3B). No cyclin D1
expression was seen in developing guard hair follicles of
cIκBαΔN mice. However, normal cyclin D1
expression was observed in vibrissae and secondary awl hairs of
cIκBαΔN mice
(Fig. 3B; data not shown).

NF-κB regulates down growth, but not initiation of primary guard
hair follicle placodes. (A) Upper panel: high-resolution light
microscopy of sagittal sections of wild-type,
cIκBαΔN (ΔN) and downless
(dl) mice. Embryos were analysed at E14.5 and E15.5. Developmental
stage of placodes is indicated in each panel (0-1/2). Lower panel shows a
schematic presentation of the first typical morphological changes observed
during hair follicle induction (see also
Paus et al., 1999). Brackets
indicate placode borders, long arrows indicate apoptosis and short arrow
indicate mitosis. Asterisks indicate attempted hair follicle formation; the
club indicates loss of structural organisation. (B) In situ
hybridization of wild-type and cIκBαΔN
(ΔN) mice at E14.5 and E15.5 using a cyclin D1 sense and antisense
probe. Vibrissae follicles also show cyclinD1 expression in dermal condensate
(wild type E14.5). s, sense probe; as, anti-sense probe; vib., vibrissae.
Stages of hair follicle development are indicated beneath placodes in
wild-type sections. Broken lines indicate the boundary between epidermis and
dermis. (C) Analysis of cell proliferation in the epidermis of E14.5
and E15.5 wild-type and IκBαΔN (ΔN) embryos. BrdU
incorporation into the DNA was detected with an anti-BrdU antibody and
subsequent peroxidase reaction (brown nuclei). There are also a few nuclei
stained in the interfollicular epidermis and dermis of wild-type and ΔN
embryos. Developmental stages are indicated in each panel. P, placode.
Brackets indicate placode borders.

BrdU incorporation revealed that proliferative cells were readily detected
from stage 0/1 on in wild-type guard hair placodes, while in areas of
attempted hair follicle formation in
cIκBαΔN mice proliferative cells were
missing (Fig. 3C). In wild-type
embryos, the proliferative cells were seen in the proximal part of placodes
where NF-κB activity was observed in κGal mice (see
Fig. 2). The above results
strongly suggest a role of NF-κB in proliferation and down growth of
guard hair placodes.

Lack of NF-κB activity leads to loss of structural organization
of the developing epidermis

HRLM and TEM analysis of E14.5 (Fig.
3A, Fig. 4A) and
E15.5 (Fig. 3A,
Fig. 4B) downless and
cIκBαΔN mice showed a severe loss of
structural organization in the epidermis at sites of placode formation when
compared with wild-type embryos at the same stage. These degenerative
processes may be the result of a lack of further placode down growth. There
was a pronounced reduction in the number and size of desmosomal junctions,
increased vacuolization of keratinocytes and increased apoptosis in the
epidermis and the underlying dermis at sites of placode formation at E14.5 in
cIκBαΔN and downless mice
(Fig. 4A, middle and lower
panels). In cell lines and some tissues, the lack of NF-κB activity,
especially in the presence of TNF signalling, is known to cause apoptosis
(Aggarwal, 2003). However, in
cIκBαΔN and downless mice,
apoptosis was mostly observed in the suprabasal layer of the epidermis and
also in the underlying dermal condensate, where NF-κB is normally not
activated. It may, therefore, be related to the general loss of structure and
attempt of reorganization in the absence of placode formation. The vacuoles
observed in the degenerating keratinocytes contained lipid-like material (see
Fig. 4A). The reason for the
vacuolization remains unknown. At E15.5, apoptosis in the suprabasal layer of
the epidermis remained or even increased, but intercellular contacts such as
desmosomes were being rebuilt, leading to less intercellular spaces than
observed at E14.5 (Fig. 4B).
This indicates that, at E15.5, the epidermis begins to regain its normal
organization.

NF-κB in vivo is downstream of Eda A1 and EdaR

Previous overexpression studies in transformed cell lines have shown that
NF-κB can be activated by Eda A1 and EdaR via the IKK pathway
(Kumar et al., 2001). It was
important to investigate whether in vivo NF-κB acts downstream of Eda
A1/EdaR. For this purpose, tabby (mutant Eda A1), downless
(mutant EdaR) and, as a control, cIκBαΔN
mice were mated into κGal reporter mice
(Fig. 5A). Embryos from these
matings did not reveal any NF-κB activity in guard hair placodes at E14
and E15 (Fig. 5A). As expected,
in cIκBαΔN × κGal
embryos, NF-κB activity was blocked, whereas in tabby or
downless × κGal embryos activity was still
present in endothelial cells of the blood vessels and other sites independent
of Eda A1/EdaR signalling (Fig.
5A, and data not shown).

In newborn mice of the same matings, NF-κB activity was also strongly
reduced (tabby and downless × κGal) or
absent (cIκBαΔN ×κ
Gal) in all secondary hair follicles
(Fig. 5B, right panel), which
did show NF-κB activity in wild-type κGal mice
(Fig. 2B). Therefore,
NF-κB is activated downstream of Eda A1/EdaR in all primary and
secondary follicles, including awls, which demonstrates that development of
awl hairs is mainly independent of NF-κB activity. However, the residual
NF-κB activity in many secondary hair placodes of tabby or
downless × κGal at P0
(Fig. 5B) supports our finding
of a more severe phenotype in cIκBαΔN
mice with regard to zigzag hair and molar tooth development, compared with
tabby mice (see Fig.
7) (Cui et al.,
2003; Ohazama et al.,
2004b). This suggests that in these ectodermal organs NF-κB
is regulated by additional signals.

The observed activity of NF-κB in guard hair placodes at E14.5
coincided with localized EdaR expression at this site. By contrast, EdaR was
still uniformly expressed in the entire epidermis in wild-type and in
cIκBαΔN mice at E13.5
(Fig. 5C), supporting previous
observations (Headon and Overbeek,
1999). Some localized EdaR expression is also observed in
cIκBαΔN mice at E14-E14.5, while at
E15.5 EdaR expression was absent (Fig.
5C). The mechanism which prevents EdaR expression in
interfollicular epidermis, instead restricting it to placodes around E14,
currently remains unknown. However, this event can still occur in
cIκBαΔN mice. The complete absence of
placodal EdaR expression at E15.5 can be interpreted in two ways. First, that
NF-κB is responsible for further EdaR upregulation. However, this
possibility is contrary to the observation that EdaR expression was normal in
awl hairs of cIκBαΔN mice at E17.5 and
P0 (Fig. 5C). Second, placodal
keratinocytes may rapidly reorient themselves to epidermal keratinocytes in
the absence of further specific placode growth signals (see
Fig. 4).

Note that in wild-type embryos, EdaR expression is located in the
downgrowing, proximal part of the placode, identical to NF-κB activity
in κGal reporter mice (see
Fig. 5C). No differences of
EdaR expression between wild-type and
cIκBαΔN mice were observed in secondary
awl hairs at E17.5 and P0, and in vibrissae follicles at any time point
(Fig. 5C). Ubiquitous
ectodermal Eda A1 expression levels in downless and
cIκBαΔN mice were also indistinguishable
from those in wild-type mice at all time points, demonstrating that Eda A1
expression does not require NF-κB
(Fig. 5C). In wild-type mice,
Eda A1 expression was typically absent from the early placodes, while Eda A1
was observed later in hair germs and peg stages, and in the hair follicle
matrix and pre-cortex (Fig. 5C,
P0). The Eda A1 mRNA probe used for this experiment recognizes all Eda A1 mRNA
isoforms.

NF-κB activity in hair placodes was further verified by using a
murine anti-sense probe of IκBα, which is a known NF-κB
target gene in cells with activated NF-κB p50/p65 complexes
(Fig. 5C)
(Le Bail et al., 1993). As
expected, in wild-type embryos, IκBα mRNA expression was strongly
upregulated in the proximal part of guard hair placodes at E14.5 and E15.5
(Fig. 5C). In
cIκBαΔN and downless embryos,
no IκBα mRNA expression was detected in the epidermis, but
upregulation was detected in vibrissae
(Fig. 5C, E14.5 and E15.5). The
lack of IκBαΔN RNA detection in
cIκBαΔN mice was presumably due to low
affinity of the mouse IκBα mRNA probe for the human
IκBαΔN RNA. However, a human IκBα mRNA probe
revealed strong IκBαΔN expression throughout the epidermis
and in secondary hair follicles of
cIκBαΔN mice, while in wild-type mice,
there was only very weak staining because of low cross-reaction with
endogenous mouse IκBα (Fig.
5C). At P0, both wild-type and
cIκBαΔN mice presented IκBα
mRNA upregulation in all secondary hair and late-stage guard follicles, and in
the basal layer of the interfollicular epidermis
(Fig. 5C, mouse
IκBα). The upregulation of IκBα mRNA in the basal
layer, and in vibrissae and awl hairs of
cIκBαΔN mice is either independent of
NF-κB activity, or the NF-κB activity in these cells is so low
that it is not detectable in the κGal mice. Thus, we have formally
proven that in vivo NF-κB acts downstream of Eda A1/EdaR. The
integration of NF-κB into the EdaA1/EdaR signalling pathway with all its
components is depicted in Fig.
8.

To investigate whether activation of NF-κB can restore placode down
growth, skin explants of tabby × κGal embryos at
E13.5 were treated with recombinant Fc-Eda A1, Fc-Eda A2, TNFα or PMA
for 24 hours (Fig. 6A). Eda A1
has previously been shown to be able to recover placode induction in
tabby mice (Mustonen et al.,
2004). Eda A2, a ligand of XEDAR (X linked ectodermal dysplasia
receptor), was used as a negative control, as it did not induce NF-κB
activity in hair follicles and cannot restore hair growth in tabby
mice (Gaide and Schneider,
2003; Mustonen et al.,
2003).

At E13.5 + 1 day (=E14.5), X-Gal staining showed that NF-κB activity
in hair placodes was only re-established in Eda A1- but not in Eda A2-treated
explants (Fig. 6A, upper
panels). The Fc-Eda A1 also induced NF-κB in surrounding keratinocytes,
because at E13.5 EdaR is still expressed uniformly in the epidermis, before it
becomes restricted to placodes at E13.5 + 1 day
(Fig. 6A, upper panels).
Endogenous Eda A1 is obviously not in its active form at E13.5 and, thus, is
not yet able to interact with EdaR to activate NF-κB. The recombinant
Fc-Eda A1, however, simulates the active form of Eda A1.

TNFα stimulated NF-κB ubiquitously, including the dermis and
some blood vessels, and, thus, placodes were not clearly distinguishable
anymore in tabby × κGal explants. However, an
antisense probe of sonic hedgehog (Shh), which is an important placode marker
(St-Jacques et al., 1998)
acting downstream of Eda A1/EdaR/NF-κB
(Fig. 6B), revealed that both
EDA A1 and TNFα strongly reactivated Shh expression and, thus, placode
formation (Fig. 6A, lower
panel). It is of significance, that TNFα does not interact with EdaR
(data not shown; P.S., unpublished). Thus, TNFα can activate NF-κB
independently of EdaR in the epidermis. PMA only faintly restored Shh
expression and placode formation, indicating that keratinocytes do not respond
strongly to phorbol esters (Fig.
6A). These results not only provide additional proof that in vivo
NF-κB is downstream of Eda A1, but demonstrate that reactivation of
NF-κB is sufficient to continue guard hair placode development.

Shh mRNA expression was absent in primary guard hair placodes of
cIκBαΔN and downless embryos at
E14.5 and E15.5, and did not appear until E17, when awl hairs develop
(Fig. 6B and data not shown).
In tabby mice, Shh expression showed the same temporal expression
pattern (Laurikkala et al.,
2002). In guard hairs of wild-type embryos Shh was first detected
at developmental stage 1-2 (Fig.
6B, E14.5 wild type), and became strongly upregulated at germ-peg
stage (stage 2-3; Fig. 6B,
E15.5 wild type), which is the stage where hair development is arrested in
Shh–/– mice
(Mill et al., 2003;
St-Jacques et al., 1998) (for
a review, see Schmidt-Ullrich and Paus,
2005). In conclusion, in guard hairs Shh expression depends
directly or indirectly on NF-κB activity and, thus, is induced
downstream of Eda A1/EdaR/NF-κB signalling. In awl hairs, Shh expression
is independent of NF-κB.

Total number of secondary hairs is reduced in
cIκBαΔN mice

We have reported previously that
cIκBαΔN mice also lack fine zigzag
underhairs and, thus, adult animals have greatly decreased numbers of hair
follicles overall (Schmidt-Ullrich et al.,
2001). At E17.5, hair numbers were reduced because of absence of
guard hairs and at P0 they only presented with about 50% of wild-type hair
numbers (Fig. 7A, upper and
lower panels). The analysis of the different stages of HF development at E17.5
and P0 supported the fact that only one hair type from the second wave (awls)
develops in cIκBαΔN mice
(Fig. 7B). However, there are
currently no mechanisms to differentiate correctly between awl, auchene or
zigzag placodes, except for the initiation time points of the second (E16,
awl/auchene) and third (E18/P0, zigzag) wave
(Philpott and Paus, 1998;
Schmidt-Ullrich and Paus,
2005) (see also Fig.
8). Thus, most stage 1 placodes at P0 are likely to give rise to
zigzag hair follicles, which do not develop until around birth, i.e. during
the third wave of hair follicle development
(Schmidt-Ullrich and Paus,
2005; Vielkind and Hardy,
1996). cIκBαΔN mice mainly
presented germ stage placodes at E17.5, and germ and peg stage placodes at P0,
while in wild-type mice, several different stages were present in an almost
equal distribution (Fig. 7B).
At P0, in cIκBαΔN mice, only very few
stage 1 placodes were detected compared with wild-type mice
(Fig. 7B). This is an
indication that in cIκBαΔN mice, zigzag
follicles may stop developing at a very early stage, similar to guard hair
follicles.

NF-κB is needed for secondary zigzag hair development, where it
may also be regulated by TROY. (A) Upper panel: cryosections of
E17.5 and P0 wild-type and cIκBαΔN
(ΔN) mice, stained with Haematoxylin/Eosin. Lower panel: hair follicle
numbers of E17.5 embryos [wild type,
cIκBαΔN (ΔN); n=3 each]
and newborn (P0; n=9
cIκBαΔN,
n=6 wild type) were counted per microscopic field (mf). Mean values were
calculated and presented in bar graphs, including standard deviations.
P values show a significant difference:
*P<0.0158 versus wild type. for E17.5, and
†P<0.0001 versus wild type for P0. At P0, the
mean value reveals 50% less hairs in
cIκBαΔN mice compared with wild type:
38/mf in cIκBαΔN versus 75/mf in wild
type. (B) The different developmental stages of hair follicle
development (stage 1, placode – early bulbous peg stage 5, indicated to
the left of the table) in wild type and
cIκBαΔN (ΔN) mice at E17.5 and P0
are presented in percentage (%) of total number of hairs. (C) In situ
hybridization of sagittal skin sections of E14.5, E15.5, E17.5 and P0 embryos
using a TROY antisense probe. Broken line indicates the boundary between
epidermis (E) and dermis (D). Arrows indicate placodes and (at P0) the matrix
of a guard hair follicle.

The more severe phenotype of cIκBαΔN
mice compared with tabby and downless mice, and our finding
that there is still some residual NF-κB activity found in secondary hair
follicles of tabby and downless × κGal
mice, implies that factors other than Eda A1/EdaR must regulate NF-κB
activity in zigzag hairs. These may include two recently discovered members of
the TNF family, e.g. XEDAR and its ligand Eda A2, and the orphan receptor
TAJ/TROY (Tnfrsf19 – Mouse Genome Informatics), the ligand of which
remains unknown (Kojima et al.,
2000; Yan et al.,
2000). Both proteins are expressed in hair follicles and in teeth,
and are known to activate NF-κB in vitro
(Kojima et al., 2000;
Ohazama et al., 2004a;
Yan et al., 2000). Although
studies with XEDAR-deficient mice revealed that XEDAR is dispensable for
ectodermal appendage development (Newton
et al., 2004), there may be redundancy between TROY and XEDAR
signalling in hair follicle development. Therefore, we analysed the expression
of TROY during the most important stages of hair development.

Interestingly, TROY mRNA was not expressed at E14, when primary guard hairs
develop, and at E15.5 only very weak staining was observed
(Fig. 7C). But strong
expression of TROY mRNA was detected at E17.5 and P0, when secondary follicles
form (Fig. 7C). Expression of
TROY co-localized with NF-κB activity (see
Fig. 2). In
cIκBαΔN mice, TROY expression was still
observed at P0, indicating that like Eda A1, TROY is not regulated by
NF-κB (data not shown). It was also expressed in the matrix of guard
hair follicles at P0 (Fig. 7C).
According to the expression pattern, one can deduce that TROY may be
specifically involved in regulating secondary hair follicle development.

DISCUSSION

We show that NF-κB is dispensable for hair placode initiation, yet it
is essential for the subsequent down growth and proliferation of hair placode
keratinocytes. In addition, we demonstrate for the first time that in vivo
NF-κB is activated downstream of Eda A1 and EdaR signalling. The
variable requirement of NF-κB for the development of each of the four
types of pelage hair follicles (see Fig.
8) underscores the emerging concept that the development of
different skin appendage subtypes is regulated by differential molecular
controls. Finally, we provide evidence that NF-κB-dependent hair placode
down growth involves downstream induction of Shh and cyclin D1 expression.

So far it has not been known at which stage hair placode development is
arrested in tabby, downless or
cIκBαΔN mice. Our finding that primary
guard hair placode development is interrupted in
cIκBαΔN and downless mice at
pre-placode stage 0/1, and that Eda A1/EdaR/NF-κB are not needed for
placode initiation, supports data from K14-Dkk1 transgenic mice, where hair
placodes fail to develop in the absence of Wnt
(Andl et al., 2002). This
indicates that Wnt is required for initiation of placode formation, and that
Eda A1/EdaR/NF-κB is most probably activated directly downstream of the
initiating Wnt signal. Furthermore, as was shown here and in a previous report
(Headon and Overbeek, 1999),
EdaR is still expressed ubiquitously in the epidermis at E13.5. Thus, there
has to be a yet unknown signal that directs EdaR expression exclusively to
hair placodes to start NF-κB signalling. Wnt is a possible candidate, as
the K14-Dkk1 transgenic mice no longer revealed placodal EdaR expression at
E14.5 (Andl et al., 2002).

Working hypothesis on the role of NF-κB in guard, zigzag or awl
hair development. The upper panel displays the degree of involvement of
NF-κB or EdaR/NF-κB during the development of the four pelage hair
types, proceeding in three different waves. Activation of NF-κB is
reflected by the X-Gal activity in the NF-κB reporter mice
(κGal). *The NF-κB activating signal for the
formation or down growth of zigzag placodes remains unknown. A possible role
of EdaR/NF-κB in the morphogenesis of guard hairs has also not been
identified yet (indicated by `?'). The lower scheme indicates the time point
at which Eda A1/EdaR/NF-κB contribute to the development of each hair
type. Shh and cyclin D1 are downstream of EdaR/NF-κB in guard hair
follicles. It remains to be determined whether this is also true for zigzag
follicles, and whether NF-κB directly regulates Shh.

The loss of structural placode organization observed in
cIκBαΔN and downless mice may
account for the previously described `delayed epidermal differentiation' in
tabby embryos at E14 and E15
(Laurikkala et al., 2002). The
apoptotic cells we observed in the epidermis of
cIκBαΔN and downless mice were
localized in the suprabasal layer and the dermis, where no NF-κB
activity was found. Therefore, we can conclude that NF-κB has no
anti-apoptotic function downstream of Eda A1/EdaR in hair forming
keratinocytes in vivo, which is in agreement with earlier results obtained
from analysing tabby teeth
(Koppinen et al., 2001).

In the current study, we asked whether NF-κB regulates keratinocyte
proliferation by direct or indirect activation of cell cycle genes. Cyclin D1
was previously described as a direct NF-κB target gene in several cell
types (Hinz et al., 1999), but
in vascular smooth muscle cells, for example, cyclin D1 is not activated by
NF-κB (Mehrhof et al.,
2005). Analysis of the temporal expression pattern of the G1 phase
regulator, cyclin D1, in placodal keratinocytes revealed that upregulation did
not take place until developmental stages 2-3, and, hence, correlated with Shh
upregulation. Thus, hair placode keratinocytes may be another example where
cyclin D1 is not directly regulated by NF-κB. Furthermore, cyclin D1
expression is entirely independent of NF-κB activity in awl hairs (data
not shown). In this hair type, Eda A1/EdaR/NF-κB is neither required for
placode formation and subsequent down growth, nor for mature follicle
development, but is needed to determine the ultrastructure of the hair.

Our data suggest that cyclin D1 is more likely to be induced by Shh and/or
Wnt10b signalling, which are both downstream of Eda A1/NF-κB activation
(R. Schmidt-Ullrich, unpublished) (Andl et
al., 2002; Laurikkala et al.,
2002). Furthermore, Shh has previously been shown to be essential
for cyclin D1 expression (Mill et al.,
2003). Although Shh expression does not appear until stage 1 of
hair follicle morphogenesis and is described as a target gene ofβ
-catenin (Gat et al.,
1998; Huelsken et al.,
2001), Eda A1/NF-κB may directly regulate epidermal Wnt10b
expression, as placodal Wnt10b expression is also absent in tabby
mice at E14.5 (Andl et al.,
2002). Because expression of cyclin D1 can already be weakly
detected in stage 1 placodes, there may also be synergy between Eda
A1/EdaR/NF-κB, Shh and Wnt signalling that results in a strong growth
signal. The identification of specific target genes of NF-κB in hair
placodes may answer this question.

Analysis of κGal × tabby mice has helped to
demonstrate the necessity of NF-κB in guard hair follicle development:
skin explants from these mice showed that classical signals inducing
NF-κB activity, although not physiologically relevant for hair placode
formation like TNFα and PMA, can regenerate placodes at E13.5 + 1 day.
These results clearly reveal NF-κB as an essential promoter of guard
hair placode growth. After TNFα, and to a lesser degree after Eda A1,
treatment of tabby × κGal explants, X-Gal
staining was also observed in interfollicular keratinocytes (see
Fig. 6). Thus, theoretically,
NF-κB can be activated anywhere in the epidermis if the right signal
appears. However, the Shh probe only marked hair placodes. Therefore, Shh is
downstream of NF-κB in those epidermal keratinocytes that previously
must have been programmed for hair placode fate in κGal ×
tabby mice at E13.5. The programming most probably includes a
generally permissive signal (i.e. `make an appendage'), which was previously
proposed to be dermal, and which may facilitate the formation of gradients of
placode activators versus inhibitors defining placode borders
(Hardy, 1992). This is in
agreement with our finding that some placode initiation processes have already
taken place in tabby, downless and
cIκBαΔN mice.

NF-κB activity and EdaR expression were detected in all hair types,
including secondary awls and vibrissae. The bona fide development of awl hairs
and most vibrissae types is independent of NF-κB activity, because it is
not affected in
cIκBαΔN,
tabby, downless or crinkled mice
(Gruneberg, 1971;
Headon et al., 2001;
Schmidt-Ullrich et al., 2001)
(see also Fig. 5A, right
panel). Hence, EdaR/NF-κB activity would not be expected in these hair
types. However, awl hairs do have a slightly abnormal shape in all these mice,
suggesting that Eda A1/EdaR/NF-κB regulate the correct ultrastructure
(see Fig. 8)
(Falconer, 1952;
Schmidt-Ullrich et al., 2001).
Moreover, mammary glands, which also exhibit strong embryonic NF-κB
activity, develop and function normally in
cIκBαΔN mice, even though nipple
morphology is altered as in tabby mice (data not shown)
(Mustonen et al., 2003). Eda
A1 and EdaR are expressed and active not only in mammals, but also in bird
feather tracts and fish scales (Houghton
et al., 2005; Kondo et al.,
2001; Pispa and Thesleff,
2003; Sharpe,
2001; Thesleff and Mikkola,
2002). In the Japanese fish medaka (Oryzias latipes), for
example, it was discovered that the rs-3 (reduced scale-3) locus encodes EdaR
(Kondo et al., 2001). Fish
with mutations in rs-3 completely lack scales
(Kondo et al., 2001). Thus, in
vertebrates, Eda A1/EdaR/NF-κB signalling is required for the
development and shaping of ectodermal appendages, which suggests that it is an
evolutionary conserved pathway. However, this appears to have become redundant
for the development of some mammalian appendages such as awl hairs, vibrissae
or mammary glands.

Complete lack of secondary zigzag hairs in
cIκBαΔN mice also points to an important
role of NF-κB in the development of this pelage hair type
(Fig. 7). In part, zigzag hair
development depends on Eda A1/EdaR signalling, because tabby and
downless mice also do not develop proper zigzag hairs
(Gruneberg, 1971;
Vielkind and Hardy, 1996). But
rescue experiments in tabby mice reveal that transgenic Eda A1
expression only reconstitutes guard and not zigzag hair follicles
(Cui et al., 2003;
Gaide and Schneider, 2003).
Furthermore, it is noteworthy that in wild-type mice, Eda A1 overexpression in
epidermal keratinocytes equally leads to the absence of zigzag hairs
(Mustonen et al., 2003). Thus,
the dose of Eda A1 signalling and its spatial distribution need to be
controlled by yet unknown factors in order to form normal zigzag hairs.
Importantly, tabby mice develop abnormal awl instead of zigzag hairs,
resulting in almost wild-type hair numbers
(Cui et al., 2003). This is in
contrast to cIκBαΔN mice and strongly
suggests that NF-κB activation downstream of Eda A1 alone is not
sufficient to develop complete zigzag hair follicles. This is also supported
by our finding of residual NF-κB activity in many secondary hair
placodes of tabby and downless × κGal
mice at P0. The orphan TNF receptor TROY was first expressed at E17, hinting
that perhaps both TROY and Eda A1/EdaR are needed to form zigzag hairs via
NF-κB (see Fig. 8). Owing
to possible redundancy, one cannot exclude that XEDAR and other NF-κB
activators are also involved. In such a scenario, residual EdaR-independent
NF-κB activity originating from TROY, XEDAR or other NF-κB
activators was predicted to occur in early secondary hair placodes of
tabby and downless mice (see
Fig. 5B).

The ability to develop awl instead of zigzag hairs in tabby mice
suggests that the initiating developmental pathways leading to both pelage
hair types are identical, depending on Noggin, Lef1, BMP and Wnt
(Botchkarev et al., 1999;
Botchkarev et al., 2002;
Jamora et al., 2003;
Schmidt-Ullrich and Paus,
2005). However, the subsequent regulation of the morphological
ultrastructure of zigzags, such as the two `kinks', seem to be regulated
specifically by Eda A1/EdaR/NF-κB (see
Fig. 8). Molar tooth
development presents yet another example where Eda A1/EdaR/NF-κB
signalling regulates the morphogenesis of cusps
(Ohazama et al., 2004b). Thus,
in ectodermal appendage development the specific role of NF-κB depends
on the type of appendage, and controls either early developmental or later
morphogenetic processes.

We have provided a comprehensive analysis of the differential functional
requirement and the spatial and temporal activation of NF-κB during
primary and secondary hair follicle development. We further presented a
genetically based dissection of the integration of NF-κB within upstream
(Eda A1, EdaR) and downstream (Shh, cyclin D1) signalling modules and genes.
This will be the basis for future analysis of gene networks that are under
direct control of NF-κB, and of the mechanisms that determine redundancy
with NF-κB-independent pathways in epidermal appendage ontogeny.

Acknowledgments

We thank Karin Ganzel and Sarah Ugowski for excellent technical help, and
Gundula Pilnitz-Stolze (Hautklinik UKE, Universität Hamburg, Hamburg,
Germany) for performing the immunohistochemistry in
Fig. 1C. The authors also thank
Joerg Huelsken for providing the downless, Shh and tabby probes; Atsushi
Ohazama (Showa University Dental School, Tokyo, Japan) for providing the TROY
probe; Gregory Shackleford for providing the Wnt10a and Wnt10b probes; and
Irma Thesleff for providing tabby mice. This work was supported in
part by a BMBF grant to C.S.

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